U.S. patent application number 10/880783 was filed with the patent office on 2005-12-29 for method of assaying denaturation of proteins.
Invention is credited to Bromberg, Steven E., Cumberland, Brendi M., Dickson, Kelly A., Gross, Adam F..
Application Number | 20050287616 10/880783 |
Document ID | / |
Family ID | 35506325 |
Filed Date | 2005-12-29 |
United States Patent
Application |
20050287616 |
Kind Code |
A1 |
Gross, Adam F. ; et
al. |
December 29, 2005 |
Method of assaying denaturation of proteins
Abstract
The present invention encompasses a method for performing an
antibody-antigen assay using an ion-exchange technique prior to
performing an antibody-antigen assay. Actives from
protein-denaturing solutions are physically removed from the test
sample, preferably by using an ion-exchange resin. The present
invention also includes a method of performing an antibody-antigen
assay, which does not use coating antibody on the sample holder.
These methods for removing or eliminating interferences can be used
in an ELISA to more effectively, efficiently and promptly screen
formulations containing protein-denaturing compounds for efficacy
against proteins in common allergens and detect and quantify such
proteins.
Inventors: |
Gross, Adam F.; (Pleasanton,
CA) ; Cumberland, Brendi M.; (Pleasanton, CA)
; Dickson, Kelly A.; (Pleasanton, CA) ; Bromberg,
Steven E.; (Pleasanton, CA) |
Correspondence
Address: |
THE CLOROX COMPANY
1221 BROADWAY PO BOX 2351
OAKLAND
CA
94623
US
|
Family ID: |
35506325 |
Appl. No.: |
10/880783 |
Filed: |
June 29, 2004 |
Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
G01N 33/5306
20130101 |
Class at
Publication: |
435/007.92 |
International
Class: |
G01N 033/53; G01N
033/542; G01N 033/537 |
Claims
What is claimed is:
1. A method of performing a protein assay in steps comprising: (a)
denaturing a protein sample to create a test sample; (b) removing
any interfering species from the test sample to create a prepared
sample; (c) adding an enzyme conjugate specific for the protein to
the prepared sample, wherein a protein-enzyme conjugate complex is
formed; and (d) measuring the amount of resulting protein-enzyme
conjugate complex
2. The method of assay according to claim 1, wherein the protein is
an antigen.
3. The method of assay according to claim 2 wherein the protein
specificity of step (c) is obtained through the use of an
antibody.
4. The method of assay according to claim 3, wherein the
interfering species is a chemical agent; and the step of removing
comprises a physical removal.
5. The method of assay according to claim 1 wherein the protein
sample is pure.
6. The method of assay according to claim 1 wherein the protein
sample is impure.
7. A method of performing an antibody-protein assay in steps
comprising: (a) denaturing a protein sample to create a test
sample; (b) removing any protein-denaturing agents from the test
sample to create a prepared sample; (c) adding the prepared sample
to a sample holder; (d) washing the sample holder to remove any
unbound antigen; (e) adding an enzyme conjugate specific for the
antigen to the prepared sample, wherein an antibody-enzyme complex
is formed; (f) removing any unbound enzyme; and (g) measuring the
amount of antibody-enzyme complex.
8. The method of performing an antibody-protein assay according to
claim 7, wherein the protein is an antigen.
9. The method of performing an antibody-protein assay according to
claim 7, wherein protein is denatured by the step of applying a
denaturing agent.
10. The method of performing an antibody-antigen assay according to
claim 9, wherein the protein-denaturing agent is a chemical.
11. The method of performing an antibody-antigen assay according to
claim 1, wherein the sample holder is a multiple well plate
allowing multiple prepared samples to be tested at the same
time.
12. The method of performing an antibody-antigen assay according to
claim 11, wherein the sample holder is coated with capture
antibody.
13. The method of performing an antibody-antigen assay according to
claim 7, wherein the step of physically removing said interfering
chemical species is performed using an ion-exchange resin.
14. The method of performing an antibody-antigen assay according to
claim 13, wherein interfering chemical species are physically
removed from the prepared sample by the step of passing the sample
through an ion-exchange resin column.
15. The method of performing an antibody-antigen assay according to
claim 13, wherein interfering chemical species are physically
removed from the prepared sample by the step of adding an
ion-exchange resin directly into the prepared sample in the sample
holder.
16. The method of performing an antibody-antigen assay according to
claim 13, wherein the ion-exchange resin is a cationic, anionic or
amphoteric resin.
17. The method of performing an antibody-antigen assay according to
claim 13, wherein the test sample is allowed to react with the
ion-exchange substance for between 5 seconds and 3 hours.
18. The method of performing an antibody-antigen assay according to
claim 13, wherein the ion-exchange resin is either a polymer-based
resin or a silica-containing material.
19. The method of performing an antibody-antigen assay according to
claim 13, wherein the ion-exchange resin is capable of removing
protein-denaturing agents generated from salts in the test
sample.
20. The method of performing an antibody-antigen assay according to
claim 13, wherein the level of said actives is less than 90% of the
prescribed resin capacity.
21. The method of performing an antibody-antigen assay according to
claim 13, wherein color, radioactive or fluorescent labeling
techniques are used to measure the amount of resulting product.
22. The method of performing an antibody-antigen assay according to
claim 21, wherein the labeling techniques include
self-amplification.
23. The method of performing an antibody-antigen assay according to
claim 10, wherein the denaturing agent comprises quaternary
ammonium compounds.
24. The method of performing an antibody-antigen assay according to
claim 23, wherein the ion-exchange resin is capable of removing
actives generated from quaternary ammonium compounds.
25. The method of performing an antibody-antigen assay according to
claim 24, wherein the quaternary ammonium compounds are about
0.001%-10% by weight of the test sample.
26. A method of performing an antibody-antigen assay in steps
comprising: (a) applying a denaturing solution to a bound protein
sample to create a test sample; (b) removing remaining denaturing
solution from solution; (c) adding detection antibodies to form an
protein-antibody complex; (d) removing unbound detection
antibodies; (e) adding an enzyme conjugate to create a prepared
sample; (f) removing any unbound enzyme; (g) adding an enzyme
substrate to generate a labeled product; and (h) measuring the
amount of resulting product.
27. The method of performing an antibody-antigen assay according to
claim 26, wherein the steps of removing are achieved by
washing.
28. The method performing an antibody-antigen assay according to
claim 26, wherein color, radioactive, or fluorescent labeling
techniques are used to measure the amount of resulting product.
29. The method of performing an antibody-antigen assay according to
claim 28, wherein the labeling techniques include
self-amplification.
30. The method of performing an antibody-antigen assay according to
claim 26, wherein the denaturing solution comprises quaternary
ammonium compounds, acids, bases, salts, oxidizers and
reductants.
31. The method of eliminating the effect of interferences according
to claim 26, wherein the quaternary ammonium compounds are about
0.001%-10% by weight of the test sample.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for assaying the
effects of protein-denaturing agents, and allows for both
qualitative and quantitative measurements of the efficacy of such
actives. In particular, the invention contemplates the use of
antibody-antigen assays, and achieves the desired result by
techniques for minimizing or eliminating interferences in
antibody-antigen assays of test samples containing
protein-denaturing compounds. Effective screening of efficacy of
protein-denaturing compounds is achieved via these methods. The
inventive methods will allow quantification of product efficacy
based on contact time with allergens. Additionally, the method
affords a means of studying the effects of protein-denaturing
compounds on various types of proteins, especially antigens. The
invention can be used in conjunction with enzyme-linked
immunosorbent assays (ELISA), as well as in other immunological
test methodologies such as radioimmunoassays, chemiluminescent
immunoassays, fluorescent immunoassays and Radioallergosorbent
tests (RAST).
BACKGROUND OF THE INVENTION
[0002] ELISA is a fundamental tool of clinical immunology that is
used as an initial screen for antigen or antibody detection. ELISAs
are highly sensitive and they have become one of the most commonly
used tests for detection and quantification of proteins in common
allergens. The ELISA method is based on measuring the amount of
binding between an antigen and an antibody specific for that
antigen and employing a label enzyme and substrate to produce an
amplified signal for analyte quantitation. By using a standard
curve, the amount of antibody-antigen binding can be transformed in
to a concentration of protein in solution. The greatest challenge
in using ELISAs to screen for efficacy of protein-denaturing
compounds/chemicals has been that the chemicals used to denature
antigen proteins also denature other proteins, including the
antibodies in an ELISA. An example of sample preparation for an
ELISA consists of mixing a candidate formula and antigen, allowing
them to react for a predetermined amount of time, then adding the
mixture to sample container (typically a well in a 96-well
microtiterplate) coated with capture antibody. Unfortunately, an
unknown amount of antibody is destroyed when adding a sample
containing a protein-denaturing formula to an ELISA.
[0003] Antigens are defined as those substances which trigger an
antibody response, and a specific class of antigens are allergens,
defined as those which trigger an allergic response. Antigens may
be proteins, haptens, small molecules such as hormones, and drugs
such as theophylline. All of these are considered to be within the
class of antigens as defined herein. Additionally, unless otherwise
clear from the context that one of these terms is intended, antigen
and allergen may be used generally interchangeably herein. Antigens
and allergens and their effects are more fully described in
co-pending U.S. patent application Ser. No. 10/817528, filed 2 Apr.
2004, commonly owned by the assignee of the present invention, the
disclosure of which is incorporated by reference herein.
[0004] Three methods have been employed to address the problem, in
a prior art ELISA, of the destruction of an unknown amount of
antibody. The first method involves chemically neutralizing the
active in the formula to prevent the destruction of the antibody.
For example, sodium sulfite can be added to an anti-allergen
formula containing oxidizing agents, such as bleach or peroxide
solutions, to neutralize any actives. Although chemical
neutralization can be effective in the context of screening for
some anti-allergen formulas, it can be ineffective in other
instances, for example, when certain salts or surfactants are used
in the anti-allergen formulas for which screening is desired.
Surfactants are advantageous in anti-allergen formulas because they
are safe for treating soft surfaces, whereas other formulas protein
denaturing agents can stain or otherwise harm soft surfaces.
[0005] Dialysis is another method for the removal of active species
that cause test interferences. This method is impractical for
testing efficacy of an anti-allergen spray or wipe because it
requires a long (as much as fifteen-hour) contact time between the
antigen and the allergen denaturing formula.
[0006] A third method utilizing an interference control may be used
to quantify the destruction of antibody. In this method, sample
containers, e.g. microtiter plate wells are coated with capture
antibody, then the test formula is added to selected wells and
allowed to incubate for the same amount of time as the antigen
would normally incubate in the well. Next, a known amount of
antigen is added to the well and the sample is run through the rest
of the ELISA. The resulting amount of antigen detected by the ELISA
from the interference assay is used as a comparison point against
which all of the other antigen level results are measured. Instead
of removing the interfering species, the interference control only
measures the maximum amount of damage the test formula causes to
the assay results. The problem with the interference method is that
some anti-allergen formulas destroy the entire coating antibody,
which produces an unreliable control sample.
[0007] Given the problems associated with immunoassaying
protein-denaturing agents, a significant need still exists for
methods that allow for both qualitative and quantitative
measurements of the efficacy of such agents. Quantification of the
efficacy of protein-denaturing agents, based upon contact time, is
not taught in the art.
[0008] There further exists a need for methods of studying the
effects of protein-denaturing compounds on various types of
proteins, especially antigens.
[0009] Moreover, the art is deficient in teaching an immunoassay
method that allows for assaying a varying purity range of proteins,
especially antigens. Thus, samples of protein, from a variety of
sources, and which may be contaminated with impurities, can
nonetheless be accurately assayed with the inventive method.
[0010] Similarly, in view of the prior art methods to remove or
adjust ELISA results to account for interferences caused by
chemicals, a significant need still exists for methods to remove
interferences or minimize their impact on ELISA testing.
SUMMARY OF THE INVENTION
[0011] The present invention provides an immunoassay method for
both qualitative evaluation and quantitative measurement of the
effects of protein-denaturing agents. "Protein denaturing" is
defined herein to include any chemical agent, or energetic source
(such as radiation) that diminishes or destroys one or more
functional sites of a protein. This includes both conformational
changes of the protein, as well as fragmentation thereof. The
present invention includes two approaches to prevent the
destruction of coating antibodies to minimize error in immunoassays
resulting from use of certain chemical agents, or energetic
sources. The first approach (the "ion-exchange" method) involves
applying a protein-denaturing solution to a protein sample to
create a test sample; physically removing any interfering chemical
species from the test sample preferably by using an ion-exchange
substance to create a prepared sample; adding the prepared sample
into a sample container (for example, a well of a microtiter plate)
coated with capture antibody; washing the sample container to
remove any unbound antigen; adding a secondary labeled antibody and
incubating for a predetermine amount of time; washing the sample
container to remove excess labeled antibody; adding an enzyme
conjugate to the prepared sample; washing the sample container to
remove any unbound enzyme conjugate; adding an enzyme substrate and
measuring the amount of resulting product. The resulting product
concentration is calculated against a standard curve.
[0012] The second approach of the present invention (the "antibody
capture" method) includes a method for reducing or eliminating the
effect of interferences in an antibody-antigen assay comprising:
coating microtiter plate wells with protein/antigen of interest;
washing the microtiter plate wells to remove any unbound antigen;
applying a denaturing solution to the protein coated wells to
create a test sample; washing the microtiter plate wells to remove
denaturing solution; adding labeled antibodies for a predetermined
amount of time; adding an enzyme conjugate to the prepared sample;
washing the microtiter plate wells to remove any unbound conjugate;
adding an enzyme substrate and measuring the amount of resulting
product against a standard curve.
[0013] Effective screening of efficacy of protein-denaturing
compounds is achieved via these methods. Allergens, especially
environmental allergens, are troublesome, and potentially harmful
to a significant proportion of the population. Domestic animals are
also sensitive to allergens, and in the case of commercial (farm)
animals, can result in economic loss. Screening permits the
development of safe and effective anti-allergen products. The
inventive methods herein also afford a means of studying the
effects of protein-denaturing compounds on various types of
proteins, especially antigens. Either approach outlined above can
be used to stop the chemical reaction between denaturing agent and
protein, thus to stop denaturation of the protein. This ability to
stop the reaction affords the ability to create a kinetic curve,
which allows quantification of the denaturing effects over time.
These methods allow quantification of product efficacy based on
contact time with allergens.
DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a depiction of a prior art sandwich ELISA test,
employing a chromogenic label, and absent any protein-denaturing
agent;
[0015] FIG. 2 is a depiction of the prior art sandwich ELISA test
of FIG. 1, demonstrating the deleterious effect of a
protein-denaturing agent upon the bound antibody;
[0016] FIG. 3 is a depiction of an antibody-capture method of the
present invention, employing a chromogenic label, and absent any
protein-denaturing agent; and
[0017] FIG. 4 is a depiction of the inventive antibody-capture
method demonstrating the undisruptive effect of a
protein-denaturing agent upon the bound antibody.
[0018] FIG. 5 is a depiction of protein-denaturing agent removal,
showing the ion-exchange step of the method of the present
invention;
[0019] FIG. 6 is a flowchart representation of a process of one
embodiment of the present invention; and
[0020] FIG. 7 is a flowchart representation of a process of a
second embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention is designed to achieve accurate
antibody-antigen assay test results with a test sample containing
protein-denaturing agents, materials, chemicals, or compounds. The
method is also suitable for assessing protein-denaturing effects of
a non-chemical nature, for example, energetic sources such as
radiation, electromagnetic fields, etc. A problem with both
qualitative and quantitative measurements of the efficacy of
protein-denaturing agents, or their effects is that they not only
denature antigens, as intended, but they also denature the coating
antibodies. When the protein denaturing solution destroys or
inactivates coating antibodies the antibodies are no longer as
effective in the ELISA test for measuring the amount of antigen in
the sample. FIG. 1 illustrates a typical prior at sandwich ELISA
10, wherein an antigen-specific coating antibody 12 is bound to a
substrate 14, to which the antigen 16 binds. A secondary antibody
18 binds to and "captures" the antigen 16. An enzyme conjugate 20
and label 22 complete the complex. FIG. 2 illustrates the adverse
effect of an active 24 upon the coating antibody 12. Here the
active 24 has denatured the antigen 16, as well as the capture
antibody 12. The rate of destruction of the antibodies may differ
from that of the antigen, and be either more or less rapid than
that of the antigen. Because of this phenomenon, ELISA test results
can show a false negative result, which makes the protein
denaturing solution appear to be less effective against the
targeted protein than it is in fact. A false negative is obtained
when the measured reduction in the protein, for example an antigen,
is less than the actual reduction thereof. Put another way, a false
negative results when a very low baseline of antigen-antibody
binding is detected.
[0022] In a first embodiment of the invention, the amount of test
interference to a prepared sample is reduced and then the prepared
sample is quantified to yield more accurate test results. The
amount of actives, which cause the test interferences, must be
reduced prior to the ELISA because in some cases so much of the
detection antibodies would be destroyed by the protein-denaturing
solution that the ELISA results would be rendered useless. This
embodiment is particularly suitable for use on chemical species
that form a salt. More specifically, these species include, but are
not limited to, quaternary ammonium compounds and surfactants, or
compounds possessing a net charge. The active species that denature
proteins in anti-allergen compositions are salts and more
specifically salts of quaternary ammonium compounds. Examples of
suitable salts and quaternary ammonium compounds that would be
appropriate for use in the present invention are discussed in
detail in Kirk-Othmer Encyclopedia of Chemical Technology 3.sup.rd,
Vol. 22, the disclosure of which is incorporated herein by
reference. The inventive methods disclosed herein permit
immunoassaying protein-denaturing actives ranging from about 0.001%
to about 10%, and are particularly effective in the 0.01 to 1%
range, all by weight of the protein-denaturing solution. The test
is thus effective over a 5 log active range, and particularly
effective over a 2 log range.
[0023] The method of the present invention utilizes physical
separation method using an ion-exchange technique to remove actives
from an ELISA test sample. Ion-exchange techniques can be carefully
chosen and used to optimize their removal actives, while still
retaining the slightly negatively charged antigen in the test
sample. The physical separation may be accomplished using
ion-exchange substances, or resins, membranes and any other similar
techniques known in the field. The ion-exchange resins may be used
in a column where the test sample is run through the column prior
to ELISA testing or the resin may be placed directly into the test
sample holders prior to ELISA testing.
[0024] The ion-exchange resin used in the present invention may be
cationic, anionic or amphoteric. Different resins must be selected
to remove different actives from the test sample. In one
embodiment, the resins are organic and polymeric, such as those
sold by the Dow Chemical Company under the trademark Dowex, and in
another embodiment, the resins are inorganic such as
aluminosilicate zeolites. Silica gel resins are one such example.
The type of resin used for the ion-exchange will determine how much
of the resin is needed to remove the actives from the test sample.
For each ion-exchange resin, the manufacturer will list the
prescribed resin capacity. The prescribed resin capacity is the
total number of chemical equivalents available for exchange per
some unit weight or unit volume of resin. The capacity may be
expressed in terms of milliequivalents per dry gram of resin or in
terms of millequivalents per milliliter of wet resin. In a
preferred embodiment of the invention, the level of actives in the
test solution is less than 90% of the prescribed resin
capacity.
[0025] The actives in the test sample are separated out using an
ion-exchange resin, resulting in a prepared sample. The prepared
sample is then placed in a sample holder. The sample holder may be
a tube or microtiter plate wells to allow for parallel processing
of many samples or any other sample holder known in the field. In
one embodiment of the invention, the sample holders are coated with
antibody specific to the antigen being tested. When the sample is
applied to the holder the coating antibody reacts with the antigen
binding the two together so that when the sample holder is
subsequently washed the antibody-antigen complex is retained in the
sample holder. Next, an enzyme conjugate specific for the antigen
is added to the prepared sample and the prepared sample is washed
once again. Finally, the amount of resulting antibody-antigen
complex is measured by adding an enzyme substrate, which indicates
the amount of resulting product by color using a chromophore or by
other radioactive or fluorescent labeling techniques. The
fluorescent or radioactive labeling may include a technique for
self-amplification.
[0026] In a second embodiment of the invention, an antibody-antigen
assay is performed without utilizing capture antibodies on the test
sample vessel. Since no capture antibody is utilized, test
interferences are eliminated, and it is therefore unnecessary to
inactivate or adjust protein-denaturing solutions or to remove the
actives from the test sample prior to an ELISA. In the method of
this embodiment, the antigen is first bound directly onto a solid
surface, such as that of a microtiter plate well, or other suitable
sample holder. Next, the protein-denaturing solution is added and
allowed to react with the antigen for a set time. The sample holder
is then washed to remove any residual product containing chemical
actives. Next, labeled, antigen specific antibodies are added to
the wells or sample vessel and allowed to form a complex. An enzyme
conjugate is added to the prepared sample and the prepared sample
is washed once again to remove excess reagent. Finally, the amount
of resulting antibody-antigen complex is measured by adding an
enzyme substrate, which indicates the amount of resulting product
by means of colorimetry. This method is also applicable when using
fluorescent or radioactive labels. This method is effective with
all classes of antibodies, and is effective when using antibody
capture ELISAs where the antibody is of the IgE class.
[0027] Since this embodiment of the invention eliminates the use of
capture antibodies, it takes significantly less time than
conventional and interference ELISAs, thus making the screening of
protein denaturing substances in solution a more rapid process. Not
only does the antibody-antigen assay method of the present
invention give more accurate and direct results but it is also
significantly more efficient than the conventional and interference
ELISA methods.
[0028] Both the first and second embodiments of the present
invention are not limited to ELISA testing for anti-allergen
compounds. The present invention relates to any methods for
minimizing or eliminating interferences in any antibody-antigen
assay for samples with protein-denaturing compounds. Although the
primary focus of this invention is on improved methods for
enzyme-linked immunosorbent assays (ELISA) these improved methods
can also be used in radioimmunoassays, fluorescent immunoassays and
Radioallergosorbent tests (RAST) or other suitable tests known in
the art, such as colorimetric tests.
[0029] FIGS. 3 and 4 illustrate an antibody-antigen assay 30 of the
present invention, wherein the antibody capture method is employed.
In FIGS. 3 and 4, a coating antibody is not used; rather an antigen
32 is bound to a substrate 34. An antigen-specific antibody 36
binds to the antigen 32, and an enzyme conjugate 38 and label 40
bind to the antibody 36 and enzyme conjugate 38, respectively.
Because the inventive method requires no coating antibody bound to
the substrate 34, there can be no destruction of coating antibody,
therefore no inherent errors in the process. FIG. 4 graphically
illustrates the limitation of the effect of an active 42 to the
target antigen 32 only. FIG. 5 is a depiction of the portion of the
ion-exchange method of the present invention wherein an
ion-exchange resin is used to physically remove a
protein-denaturing agent from a mixture containing test antigen. In
the Fig., the process 50 includes the step of contacting antigen 60
with a protein-denaturing agent 62. This results in a mixture of
some active antigen 60, denaturing agent 62, and some denatured or
inactivated antigen 64. The mixture is then passed through an
ion-exchange column 66, containing suitable ion-exchange resin 68
as known to the art. Eluting from the column 66 is a mixture of
active antigen 60, and denatured or inactivated antigen 64, which
is then incubated in a sample holder 70 containing a plurality of
coating antibody 72. The assay is concluded as described
herein.
[0030] It should be noted that ion-exchange resins can be selected
to either bind protein (antigen) and elute protein-denaturing
agent, or can bind protein-denaturing agent and elute protein. In
particular, with the latter, some ion exchange resins will bind
both protein and denaturing agent, but protein can be freed from
the resin (retaining denaturing agent) by eluting with a buffered
solution. Since a protein's net charge is influenced by pH,
changing the pH results in a change in charge, allowing the protein
to elute. An uncharged denaturing agent will exhibit pH independent
interactions with the resin.
[0031] Both the ion exchange and antibody capture methods allow
control of reaction time. For the ion-exchange method, it is
possible to use ion-exchange columns that will extract all, or
substantially all, of the protein-denaturing active in less than 30
seconds, or in less than 20 seconds, or in less than 10 seconds, or
in less than 5 seconds. Other ion-exchange columns will extract
all, or substantially all, of the protein-denaturing active in less
than 3 hours, or in less than 2 hours, or in less than 1 hour. Once
the antigen/active solution is drawn through the ion-exchange
column, all active will be removed and the reaction will be
stopped. This step can be done any time after mixing the antigen
and active. For the antibody capture method, washing the plate at
any time after the active is added stops the reaction. Both methods
allow short contact times between active and antigen, which was
previously not possible for salt and surfactant based actives.
[0032] Control of reaction times affords a variety of benefits.
When screening protein-denaturing agents for applications in
surface cleaning products, the experimenter can mimic drying times'
actual use, for example, in consumers' homes. Additionally, kinetic
curves can be created that show both the effect of concentration on
denaturation, and also the effect of time. This permits determining
a minimum reaction and a minimum drying time required to inactivate
a particular allergen/antigen, which relates to product efficacy.
Furthermore, kinetic curves can be fitted to kinetic rate laws,
which allow mathematical predictions of the effect of concentration
and elapsed time on efficacy.
Experimental Results
[0033] To assess the efficacy of the methods of the present
invention, a series of experiments were first done to quantify the
interference caused by protein-denaturing compounds in a prior art
ELISA. Since the highly destructive actives that cause the
interference can also lead to false negative results, test samples
were run through a column of ion-exchange resin to reduce the
amount of actives prior to performing the ELISA tests. Since the
ion-exchange resin also retains some of the allergen protein of
interest, tests were performed to determine the amount of allergen
lost from the test samples. Using only the necessary amount of
resin can optimize the process because the less resin used in the
ion-exchange process the less allergen protein of interest that is
retained in the resin, thus leading to great improvements in the
accuracy of the tests.
[0034] Table I below shows the effects of various surfactant-based
protein-denaturing actives on a Der p1 allergen, utilizing the
ion-exchange resins method of the present invention.
1TABLE 1 Effect of Ion-Exchange Resin in Removal of Actives
Allergen Level in Allergen Level in Active Solution Active Solution
with Passed Through Der p1 Then Passed Percent Resin Then Mixed
Through Resin Reduction of Active with Der p1 (ng/ml) (ng/ml)
Allergen Bardac 2050 138 5 93% Lonzabac 12 161 26 69% Bardac 2050 +
120 5 92% Lonzabac 12
[0035] Once the effect of the ion-exchange resin on the allergen
protein was determined, tests were performed to determine the
amount of reduction in actives by the ion-exchange resin. Samples
of active solutions diluted with water were prepared using 0.4%
Bardac 2050 by weight, 0.15% Lonzabac 12 by weight, and 0.4% Bardac
2050 plus 0.15% Lonzabac 12 by weight. These chemicals are all sold
by Lonza, Inc., Fairlawn, N.J. The prepared active solutions were
run through a column of Dowex MR-3 resin. For one set of active
test samples, Der p1 allergen (a commercially-available recombinant
protein of the Indoor Biotechnologies company) was added after
passing the solution through the ion-exchange resin. For the second
set of active test samples, Der p1 allergen was added prior to
passing the samples through the ion-exchange resin. Fourier
Transform Infrared Spectroscopy (FTIR) showed that less than 1% of
the original Bardac 2050 and less than 7% of the original Lonzabac
12 remained after passing the active test samples through the ion-
exchange resin. Using the inventive method of minimizing the
interference effect of the actives, and by taking into account the
allergen retained in the resin, the percentage reduction in
allergen caused by the actives could be accurately measured, as
shown in Table I. Allergens were measured by optical density at a
single wavelength, and concentrations were determined by
calculating against a standard curve using a Molecular Devices
SOFTmax PRO (version 3.1), and a percentage reduction was generated
by Equation 1: 1 [ control - test sample ] [ control ] .times. 100
= % reduction ( 1 )
[0036] Table II below demonstrates the accuracy improvement in
measuring actual allergen inactivation (on a Der p1 allergen) using
the ion- exchange method of the present invention compared with the
prior art interference method. In other respects, the experimental
methods were as described for Table I.
2TABLE II Comparison of Methods Percent Allergen Reduction Measured
By Percent Allergen Reduction Prior Art Interference Measured By
Ion- Active Control Exchange Method Bardac 2050 47% 93% Lonzabac 12
0% 69% Bardac 2050 + 74% 92% Lonzabac 12
[0037] For comparison purposes, ELISAs were run with interference
controls and with an ion-exchange step prior to the ELISA. The
interference control was prepared by allowing the active test
solution to be added to the sample holder for the same amount of
time that the antigen sample and then a known amount of antigen is
added and an ELISA is performed. The following is a more detailed
description of a specific interference control ELISA of the prior
art:
[0038] Capture monoclonal antibodies are bound to polystyrene
surface, and allowed to remain overnight. Excess capture antibodies
are washed with a wash buffer using a microtiter plate washer. The
protein-denaturing agent and capture antibodies are incubated for 1
hour prior to ELISA testing. This is used to evaluate the damage
caused to the antibodies by the agent. The protein-denaturing agent
is then washed off using a microtiter plate washer.
[0039] Standards and controls are prepared. Protein-denaturing
agent test samples are prepared by incubating with antigen for a
pre-determined period of time. An antigen solution of the same
concentration as that used with the product is prepared so that it
is added to the interference control wells to assist in evaluating
damage to the antibodies.
[0040] Standards, Interference control antigen solution, antigen
controls, product blanks and products are incubated in parallel for
one hour. Excess antigen from all wells is washed off using a
microtiter plate washer. A detection antibody, specific to the
antigen tested, is incubated for one hour at room temperature, and
the excess detection antibody is washed off. An enzyme conjugate is
added and incubated at room temperature for a specified time, then
excess conjugate is washed off.
[0041] A substrate such as ABTS (2,2'-AZINO-bis
(3-Ethylbenzthiazoline-6-s- ulfonic acid) or TMB
(3,3',5,5'-Tetramethylbenzidine) is incubated for a specified
period of time to develop the assay. Finally, the ELISA is read
using a microtiter plate reader such as the Spectramax 250 by
Molecular Devices.
[0042] The comparison of methods in Table 2 shows higher
percentages for the ion-exchange method indicating that it is more
effective than the interference control method at accurately
measuring allergen inactivation. The effectiveness of the
ion-exchange method is particularly apparent in the case of the
Lonzabac 12 sample, because the interference control gave a false
negative result. This false negative result happens when the
actives destroy all of the coating antibody.
[0043] In contrast to the interference control method, the
ion-exchange method was able to measure the effects of Lonzabac 12
on the allergen. Therefore, the inventive method demonstrates an
improvement in accuracy over previously available tests.
[0044] Table III below is a comparison of three different
commercially-available protein-denaturing agents on the Der p1
allergen, measured by the antibody capture test of the present
invention. The antibody capture test was conducted as follows:
microtiter plate wells were coated with the Der p1 antigen. The
microtiter plate wells were washed to remove any unbound antigen,
and a denaturing solution was applied to the protein coated wells
to create a test sample. The microtiter plate wells containing test
sample were washed to remove denaturing solution, labeled
antibodies were added for a predetermined amount of time then an
enzyme conjugate was added to the prepared sample. Next, the
microtiter plate wells were washed to remove any unbound conjugate,
an enzyme substrate was added and the amount of resulting product
was measured against a standard curve, as described in connection
with Table I.
[0045] More detail regarding the effects of the dilute (85 ppm
available chloride) and the ultra dilute (8.5 ppm available
chloride) hypochlorite solution used in the test of table III may
be found in copending U.S. patent applications Ser. No. 10/806522,
filed 23 Mar. 2004, and Ser. No. 10/828571, filed 20 Apr. 2004,
both of which are commonly owned by the assignee of the present
invention, the disclosures of which are incorporated by reference
herein.
3TABLE III Effect of protein-denaturing actives on allergen protein
Protein Concentration Protein applied in concentration After %
Reduction Sample ELISA wells active treatment of Allergen DDI Water
+ 2.8 ug/mL 2.8 ug/mL 0% Surfactant and fragrance Dilute NaOCl 2.8
ug/mL 0.016 ug/mL 99% Bleach Solution Ultra Dilute 7.2 ug/mL 3.8
ug/mL 47% NaOCl Bleach solution
[0046] Table IV below is a comparison of assay time between the
prior art neutralization and interference methods, and the antibody
capture method of the present invention. The neutralization method
employed thiosulfate as a neutralizing agent. The prior art
interference method was conducted as described for Table II above.
The antibody capture method of the present invention was as
described in reference to Table III above. Table IV illustrates the
assay time savings of the methods of the present invention.
4TABLE IV Comparison of product Efficacy Screening Prior Art Prior
Art Neutralization Interference Antibody Capture ELISA ELISA ELISA
% Allergen % Allergen % Allergen Reduction Reduction Reduction
>99% >99% >99% 3-4 hours assay 5-6 hours assay 2 hours
assay time.sup.(1) time.sup.(1) time.sup.(1) .sup.(1)Time of assay
excludes overnight coating of antigen or antibody.
[0047] The testing methods described are not intended to limit in
any manner the scope or equivalents to which the invention is
entitled, the invention is further characterized by the claims,
which follow.
* * * * *